Non-typhoidal Salmonella, including serotype Typhimurium (STm) are food-borne bacterial pathogens that cause ~1.4 million cases of diarrheal disease annually in the United States and hundreds of millions of cases worldwide. In the intestine, STm induces a strong neutrophilic inflammatory response and the production of antimicobial compounds by intestinal epithelial cells. In the face of this inflammatory response the numbers of STm in the intestine increase sharply, while the intestinal microflora of the host are dramatically reduced. The objective of this proposal is to identify the bacterial factors that allow STm to resist being killed by antimicrobials produced by the intestinal epithelium. The calf is the natural model of diarrheal STm infection that is most similar to human disease in clinical signs, host responses, and intestinal pathology. We have developed a forward genetic system to make screening for mutants feasible in ligated ileal loops in calves, a model developed at Texas A&M. We have generated a collection of over 1000 targeted deletion strains in STm, including mutants in all "Salmonella-specific" genes and we have developed microarray-based methods to screen this entire collection of mutants as a single pool. We have already confirmed the use of this system for forward genetic screening in animal infection. In AIM-1 we will screen the mutant pool in calf ligated ileal loops to identify mutants sensitive to intestinal epithelial cell-derived antimicrobials. In AIM-2 we will verify and complement these mutants in competitive infections in ligated ileal loops in calves. In AIM-3 we will determine which of these genes are important for resistance to the purified antimicrobials calprotectin and enteric 2-defensin, antimicrobials known to be produced by the intestinal epithelium during inflammation. This project is a first step toward a comprehensive determination of the molecular mechanism of the bacterial genes involved in the response to inflammation in the gut. PUBLIC HEALTH RELEVANCE: Salmonella is a leading cause of food borne illness, causing ~1.4 million cases of diarrheal disease per year and is the single most common cause of death from food-borne illnesses associated with viruses, parasites or bacteria in the US primarily in immunocompromised persons. The genes and mechanisms used by non-typhoidal Salmonellae to survive in the face of a host inflammatory response in the intestine are not well understood. These mechanisms allow Salmonellae to establish a niche in the intestine during infection, and they are shed from this niche in fecal material from infected persons and livestock continuing the cycle of transmission. Development of a better understanding of the genes and mechanisms involved in this important stage of infection is critical to breaking the cycle of transmission of this organism. This work will have a direct impact on public health.